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Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis.

Maiato H, Rieder CL, Khodjakov A - J. Cell Biol. (2004)

Bottom Line: This poleward transport results in chromosome bi-orientation and congression.Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores.However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany 12201, USA.

ABSTRACT
It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

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Related in: MedlinePlus

Spindle formation in Drosophila S2 cells stably expressing GFP/α-tubulin. Selected frames from fluorescence microscopy time-lapse sequences of mitotic spindle formation in S2 cells. (A) Spindle formation in a cell with centrosomes completely separated before NEB. Note the rapid penetration of astral MTs inside the nucleus and the process of maturation of the K-fibers. (B) Spindle formation in a cell with centrosomes only partially separated by NEB. Note that formation of K-fibers is initially seen only on the side of chromosomes that face the centrosomes. However, within just a few minutes (compare 6:00 with 11:30), prominent K-fibers appear in association with those kinetochores that are oriented away from the centrosomes. These K-fibers converge and form an ectopic pole (20:30–26:00). However, over time, all distal K-fibers become incorporated into the main spindle that forms between the centrosomes (31:30–55:30). Time is in min:s. Bars, 5 μm.
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fig1: Spindle formation in Drosophila S2 cells stably expressing GFP/α-tubulin. Selected frames from fluorescence microscopy time-lapse sequences of mitotic spindle formation in S2 cells. (A) Spindle formation in a cell with centrosomes completely separated before NEB. Note the rapid penetration of astral MTs inside the nucleus and the process of maturation of the K-fibers. (B) Spindle formation in a cell with centrosomes only partially separated by NEB. Note that formation of K-fibers is initially seen only on the side of chromosomes that face the centrosomes. However, within just a few minutes (compare 6:00 with 11:30), prominent K-fibers appear in association with those kinetochores that are oriented away from the centrosomes. These K-fibers converge and form an ectopic pole (20:30–26:00). However, over time, all distal K-fibers become incorporated into the main spindle that forms between the centrosomes (31:30–55:30). Time is in min:s. Bars, 5 μm.

Mentions: A common feature of mitosis in animals is that the two centrosomes can be at different stages of separation at the time of NEB (for review see Rieder, 1991). However, regardless of the relative positions of the centrosomes at NEB, a normal bipolar spindle ultimately forms. As originally reported (Goshima and Vale, 2003), Drosophila S2 cells used in this study frequently contain more than two centrosomes during prophase. These centrosomes are easily detectable in live cells as they generate pronounced MT asters. This feature allowed us to select cells with only two asters at NEB for our time-lapse recordings. Among 14 cells followed through mitosis, six had centrosomes positioned on opposite sides of the nucleus at NEB (Fig. 1 A). By contrast, in the other eight cells NEB occurred while the centrosomes were still relatively close to one another (Fig. 1 B).


Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis.

Maiato H, Rieder CL, Khodjakov A - J. Cell Biol. (2004)

Spindle formation in Drosophila S2 cells stably expressing GFP/α-tubulin. Selected frames from fluorescence microscopy time-lapse sequences of mitotic spindle formation in S2 cells. (A) Spindle formation in a cell with centrosomes completely separated before NEB. Note the rapid penetration of astral MTs inside the nucleus and the process of maturation of the K-fibers. (B) Spindle formation in a cell with centrosomes only partially separated by NEB. Note that formation of K-fibers is initially seen only on the side of chromosomes that face the centrosomes. However, within just a few minutes (compare 6:00 with 11:30), prominent K-fibers appear in association with those kinetochores that are oriented away from the centrosomes. These K-fibers converge and form an ectopic pole (20:30–26:00). However, over time, all distal K-fibers become incorporated into the main spindle that forms between the centrosomes (31:30–55:30). Time is in min:s. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172442&req=5

fig1: Spindle formation in Drosophila S2 cells stably expressing GFP/α-tubulin. Selected frames from fluorescence microscopy time-lapse sequences of mitotic spindle formation in S2 cells. (A) Spindle formation in a cell with centrosomes completely separated before NEB. Note the rapid penetration of astral MTs inside the nucleus and the process of maturation of the K-fibers. (B) Spindle formation in a cell with centrosomes only partially separated by NEB. Note that formation of K-fibers is initially seen only on the side of chromosomes that face the centrosomes. However, within just a few minutes (compare 6:00 with 11:30), prominent K-fibers appear in association with those kinetochores that are oriented away from the centrosomes. These K-fibers converge and form an ectopic pole (20:30–26:00). However, over time, all distal K-fibers become incorporated into the main spindle that forms between the centrosomes (31:30–55:30). Time is in min:s. Bars, 5 μm.
Mentions: A common feature of mitosis in animals is that the two centrosomes can be at different stages of separation at the time of NEB (for review see Rieder, 1991). However, regardless of the relative positions of the centrosomes at NEB, a normal bipolar spindle ultimately forms. As originally reported (Goshima and Vale, 2003), Drosophila S2 cells used in this study frequently contain more than two centrosomes during prophase. These centrosomes are easily detectable in live cells as they generate pronounced MT asters. This feature allowed us to select cells with only two asters at NEB for our time-lapse recordings. Among 14 cells followed through mitosis, six had centrosomes positioned on opposite sides of the nucleus at NEB (Fig. 1 A). By contrast, in the other eight cells NEB occurred while the centrosomes were still relatively close to one another (Fig. 1 B).

Bottom Line: This poleward transport results in chromosome bi-orientation and congression.Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores.However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, Albany 12201, USA.

ABSTRACT
It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

Show MeSH
Related in: MedlinePlus